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Unravelling the role of lysine acetylation in the regulation of glycolysis in cancer cells through the development of synthetic biology-based tools

Periodic Reporting for period 3 - AcetyLys (Unravelling the role of lysine acetylation in the regulation of glycolysis in cancer cells through the development of synthetic biology-based tools)

Reporting period: 2019-07-01 to 2020-12-31

Proteins are linear chains (polymers) of amino acids, and often viewed as the “workhorse” molecules of life. In all known organism on earth, proteins take part in essentially every process and structure. Remarkably, this diversity is achieved using only 20 different amino acids. That is, beside few rare examples, all proteins are composed of the same 20 different building blocks. However, proteins in living cells are often modified by the attachment of chemical groups to specific amino acids. In principal, these modification alter the chemical nature of the modified amino acids and by doing so may alter the structure and function of the protein. One such modification is called acetylation, whereby an acetyl chemical group is attached to the amino acid lysine. Acetylation was first identified in the 1960 on a small group of proteins, but in recent years it became clear that acetylation is widespread modification that can be found on over 2000 proteins. In particular, most of the proteins involved in metabolic processes were found to be acetylated. This observation suggests that acetylation is a mechanism for controlling cellular metabolism. Moreover, calorie restricted diet affects the level of protein modification by acetylation and may delay the onset of age related diseases. However, the role of acetylation in the regulation of metabolism is not clear yet.
In the 1920’s the German scientist Otto Warburg first suggested the link between cancer development and metabolism. In the following years his work was developed into what is currently termed the Warburg effect – the altered metabolism of cancer cells. One of the characteristics of cancer cell metabolism is high rate of glycolysis, the chain of reactions by which living cells metabolize glucose. The metabolism of glucose is executed by 10 different proteins (or enzymes), all of which were found to be acetylated.
The overall objective of our project is to determine if and how acetylation affects the metabolism of glucose and if the effect is different between normal and cancerous cells. In our study we develop tools from the field of synthetic biology that will allow us, and other research groups, to study the direct effect of protein modification by acetylation on cellular metabolism. Better understanding of the correlation between caloric intake, the level of protein modification by acetylation and the regulation of metabolism can aid in understanding the mechanism of numerous human metabolic-related human diseases such as obesity and cancer.
We have developed the molecular tools that enable to routine study of acetylated proteins in cultured mammalian cells. The study now includes three glycolytic enzymes and one non-glycolytic enzyme. We studied over ten acetylation sites on each enzyme, and identified modifications that inhibit or increase the catalytic activity of the modified enzyme. Using x-ray crystallography we determined the three-dimensional structure of the modified enzymes in order to understand the mechanism by which acetylation regulate its activity. We have also developed tools that allow us to study the reverse process – the removal of the chemical modification (termed de-acetylation). Using this and other tools we now monitor the process of de-acetylation under different conditions. This part of our work can aid the development of new generation of drugs the inhibit the rate of de-acetylation.
We have developed new tools for measuring the direct effect of specific acetylation events on protein function in cultured mammalian cells. Using these tools we have identified over a dozen of acetylation sites that significantly affect the activity of the modified protein. This information was previously unknown. We have also determined the structure of some of the acetylated proteins and thereby identified new mechanisms for metabolic regulation by acetylation.